The long-range goal of this proposal remains an understanding of prolactin action on the ovaries of higher mammals. We have shown that granulosa cells from porcine ovaries contain prolactin receptors and that these receptors are under control of hormonal and developmental influences in vivo and in vitro. In addition to prolactin receptors, there are important intracellular control points which regulate the action of prolactin on these cells. Despite comparable receptor levels, immature granulosa cells are inhibited whereas luteinizing granulosa cells are stimulated by prolactin. Estrogens seem critical to prolactin action in both cell-types, reversing the inhibitory effect and enhancing the stimulatory effect of prolactin. We will continue to probe the biochemistry of these prolactin actions, emphasizing the prolactin-estradiol interaction through studies of prolactin modulation of estrogen receptors and estrogen-mediated biochemical effects. Specifically, we will delineate the effects of prolactin, estrogen, and their interaction on cholesterol transport, metabolism, and utilization for steroidogenesis as well as the cyclic AMP/protein kinase system and the steroidogenic enzymes. Later in the grant period, we will study these questions in more refined preparations of follicular cells and ascertain the influence of follicular atresia on these hormonal effects. These data will be supplemented by studies in intact porcine follicles in which the interrelationship of steroid secretion and action can be studied more physiologically. In this latter phase of the work, we will study androgen and estrogen secretion and make estimates of steroidogenic enzymes and polypeptide receptors by cytochemistry and autoradiography, respectively, in addition to the endpoints above. This project may develop new insights into the pathophysiology of the galactorrhea-amenorrhea syndrome, a common cause of anovulation and infertility in humans.